Abstract
Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, β-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na+ or K+ currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.
Highlights
Mesenchymal stem cells (MSC) from bone marrow stroma have been broadly studied for use in the therapy of neurological disorders
The former tried to differentiate MSC using molecules involved in neural development [6], while the latter proposed that neural induction could be performed by the addition of specific chemical compounds to the culture medium (b-mercaptoethanol [BME], dimethyl sulfoxide [DMSO] and butylated hydroxyanizole [BHA]) [9]
MSC Culture Primary adult rat bone marrow MSC were isolated according to the method of Azizi et al [17]
Summary
Mesenchymal stem cells (MSC) from bone marrow stroma have been broadly studied for use in the therapy of neurological disorders. In vitro experiments seeking the ideal conditions to obtain neural cells from MSC were initially conducted by two main research groups: Sanchez-Ramos et al [6] and Woodbury et al [9]. The former tried to differentiate MSC using molecules involved in neural development (growth factors, neurotrophins, cytokines and retinoic acid) [6], while the latter proposed that neural induction could be performed by the addition of specific chemical compounds to the culture medium (b-mercaptoethanol [BME], dimethyl sulfoxide [DMSO] and butylated hydroxyanizole [BHA]) [9]
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